The production of polypeptides derived from natural sources can be limited by the expense of purification or suffer from limits on availability of starting materials. Heterologous polypeptide production is an alternative to the recovery of polypeptides from natural sources and can be used for the production of polypeptides in economically relevant amounts that are suitable for a variety of applications. Exemplary polypeptides suitable for production in heterologous expression systems include, but are not limited to, antibodies, hormones, cytokines, interleukins, enzymes, blood factors, pesticides and vaccines. Production of genetically engineered vaccine antigens, therapeutic polypeptides, industrial enzymes, biopolymers, and bioremediation agents constitute a multibillion dollar-per-year industry.
Current in vivo platforms for the production of recombinant polypeptides are limited to a relatively small number of cell-based systems that employ bacteria, fungi, insect and mammalian cells. Although bacteria can offer high yield and low-cost alternatives for production of mammalian polypeptides, cell culture systems based on higher organisms (e.g., insect or mammalian cells) generally provide polypeptides having greater fidelity to the natural polypeptides in terms of polypeptide folding and/or post-translational processing (e.g., glycosylation). Whole transgenic plants and animals have also been harnessed for the production of recombinant polypeptides, but the long development time from gene to final product can be a major drawback with these multicellular organisms, purification of the recombinant polypeptides can be difficult, and yield may be low.
Recombinant gene expression in microbial systems generally relies on one of two methods for expression construct maintenance in the host cell: episomal or integrative-based expression. Episomal expression vectors can contain the genetic elements required for recombinant gene expression (i.e., promoters, terminators, etc.) and can be maintained as independent genetic elements, usually based on a dominant drug selection system, or with a recessive auxotrophic selection system. Removal of the selection system often results in loss of the transgenic expression element as cell culture routinely confers a competitive growth advantage to cells that have lost recombinant DNA constructs. Integrative expression vectors can be more genetically stable than episomal expression vectors since they are generally maintained at specific loci in a host cell's chromosomes. This latter approach, however, can suffer from limited copy numbers of the transgenes compared to episomal strategies.
Recombinant gene expression in ciliated protists is facilitated via a variety of available expression vectors encompassing all the machinery required for transgene expression. These vectors can be incorporated as either episomal or integrative genetic elements. For example, traditional methods for the production of transgene-encoded polypeptides in ciliates such as Tetrahymena have been based on incorporation of expression cassettes into somatic loci on the macronuclear chromosomes. Although some recombinant gene expression methods employed in Tetrahymena take advantage of the high-level amplification of ribosomal DNA copy number that occurs following sexual conjugation, current methods can result in the formation of a recombinant palindrome over several generations of propagation. This can result in loss of the transgene and, consequently, in loss of expression of any polypeptides encoded by the transgene.
Increasing yield and maintaining genetic stability is important in expression system for many reasons including, but not limited to, reduced production costs. Thus, there is a need for improved methods for recombinant polypeptide production in ciliates. This invention addresses this need.